Human stool samples were filtered through a cell strainer after being diluted in 10 ml of PBS for 24 h. To separate EVs from stool and urine samples, EVs in samples were isolated by centrifugation at 10,000 × g for 10 min at 4 °C. After centrifugation, the pellet of stool samples contained bacterial cells, and the supernatant of stool and urine samples contained EVs. Bacteria and foreign particles were eliminated from stool and urine sample supernatants by sterilizing the supernatant through a 0.22 µm filter. To extract DNA from bacterial cells and bacterial EVs, bacteria and EVs were boiled for 40 min at 100 °C. To eliminate remaining floating particles and waste, the supernatant was collected after 30 min of centrifugation at 13,000 rpm at 4 °C. DNA was extracted using a DNA isolation kit (PowerSoil DNA Isolation Kit, MO BIO, USA) following the standard protocol in the kit guide. The DNA extracted from the bacterial cells and EVs contained in each sample was quantified using a QIAxpert system (QIAGEN, Germany).
Qiaxpert system
Manufactured by Qiagen
Sourced in Germany, United States
The QIAxpert system is a compact and fully automated nucleic acid extraction and purification instrument. It is designed to extract and purify DNA, RNA, or viral nucleic acids from a variety of sample types. The QIAxpert system utilizes QIAGEN's proven technologies to provide reliable and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy powersoil kit, by Qiagen (12 mentions)
Powersoil dna isolation kit, by Qiagen (7 mentions)
Ml008 01, by Welgene (6 mentions)
Dneasy blood tissue kit, by Qiagen (3 mentions)
Mobio powersoil dna isolation kit, by Qiagen (3 mentions)
Miseq system guide, by Illumina (3 mentions)
Qiacube, by Qiagen (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
45 ti rotor, by Beckman Coulter (2 mentions)
Genetitan multi channel instrument, by Thermo Fisher Scientific (1 mentions)
Qiaxpert, by Qiagen (1 mentions)
Phosphate buffered saline (pbs), by Welgene (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Nanodrop nd 1000, by Qiagen (1 mentions)
Truseq stranded mrna kit, by Illumina (1 mentions)
Medimachine, by BD (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Iscript advanced cdna synthesis kit for rt pcr, by Bio-Rad (1 mentions)
Vacutainer, by BD (1 mentions)
60 protocols using qiaxpert system
1
Stool and Urine EV DNA Extraction
2
Bacterial EVs Isolation and DNA Extraction
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Bacterial EVs were isolated from the feces using a previously described procedure20 (link). Briefly, the fecal samples were filtered through a cell strainer after being diluted in 10 mL of PBS for 24 h. EVs in fecal samples were isolated using differential centrifugation at 10,000 × g for 10 min at 4 °C. DNA from EVs was extracted as previously described21 (link). Tissue DNA extraction was performed immediately after the sample was cut without boiling or centrifuging. DNA was extracted using a DNA isolation kit (PowerSoil DNA Isolation Kit, MO BIO, USA) following the manufacturer’s instructions. Isolated DNA from each sample was quantified using the QIAxpert system (QIAGEN, Germany).
3
Isolation and Characterization of Bacterial Extracellular Vesicles
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Bacterial EVs were isolated from the sera as described previously [21 (link)22 (link)23 (link)]. Male Tg-APPswe/PS1dE9 mice were sacrificed at the age of 8 months and blood was collected from the hearts of sacrificed mice. Collected blood was centrifuged at 1,500 × g for 15 min at 4℃, and sera were separated, frozen and stored at −70℃ until use.
The sera were diluted 1 in 3 with 1x PBS (pH 7.4; ML008-01, Welgene Inc., Gyeongsan, Korea) and centrifuged at 10,000 × g for 1 min at 4℃. Then, the supernatants were collected and filtered through a 0.22-µm filter to remove bacteria and foreign particles. The separated bacterial EVs were boiled at 100℃ for 40 min. They were then centrifuged at 13,000 rpm for 30 min at 4℃, and the supernatants were collected. Bacterial DNA was extracted from the boiled EVs with a PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA USA) in accordance with the manufacturer's instructions. The DNA from the EVs in each sample was quantified with a QIAxpert system (QIAGEN, Hilden, Germany).
The sera were diluted 1 in 3 with 1x PBS (pH 7.4; ML008-01, Welgene Inc., Gyeongsan, Korea) and centrifuged at 10,000 × g for 1 min at 4℃. Then, the supernatants were collected and filtered through a 0.22-µm filter to remove bacteria and foreign particles. The separated bacterial EVs were boiled at 100℃ for 40 min. They were then centrifuged at 13,000 rpm for 30 min at 4℃, and the supernatants were collected. Bacterial DNA was extracted from the boiled EVs with a PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA USA) in accordance with the manufacturer's instructions. The DNA from the EVs in each sample was quantified with a QIAxpert system (QIAGEN, Hilden, Germany).
4
Extracellular Vesicle DNA Isolation
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To isolate EVs from human serum, whole blood was collected into serum separator tubes that were centrifuged at 3,000 × g at 4 °C for 15 min. We used 100 μL of serum from each participant. The supernatant was transferred into a new tube and centrifuged again (10,000 × g at 4 °C for 1 min) to remove floating particles. Next, the isolated serum was diluted tenfold with sterile phosphate-buffered saline, and bacteria and foreign particles were eliminated from the mixture by 0.22 μm filtration.
To extract DNA from the EVs, serum mixtures containing EVs were boiled at 100 °C for 40 min. After centrifugation (13,000 × g at 4 °C for 30 min), DNA was extracted from the supernatant free of floating particles by using the PowerSoil DNA Isolation Kit (MO BIO, USA), according to the manufacturer’s instructions. Finally, the extracted DNA was quantified using the QiAxpert system (QIAGEN, Germany).
To extract DNA from the EVs, serum mixtures containing EVs were boiled at 100 °C for 40 min. After centrifugation (13,000 × g at 4 °C for 30 min), DNA was extracted from the supernatant free of floating particles by using the PowerSoil DNA Isolation Kit (MO BIO, USA), according to the manufacturer’s instructions. Finally, the extracted DNA was quantified using the QiAxpert system (QIAGEN, Germany).
5
Genetic profiling of COVID-19 patient outcomes
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DNA from blood samples was isolated using John's method [93 (link)]. Quantity of extracted DNA was estimated using DNA High sensitivity assay kit on Qubit fluorimeter v 4.0 (Thermo Fisher Scientific). Quality of extracted DNA was assessed using agarose gel electrophoresis and QIAxpert system (QIAGEN). For genotyping, we used Axiom™ Precision Medicine Diversity Array (PMDA) Plus Kit, 96-format containing 8,68,298 markers selected for high genomic coverage (Thermo Fisher Scientific) on GeneTitan Multi-Channel (MC) Instrument (Thermo Fisher Scientific). Best markers were selected using Axiom Analysis Suite following the best practices workflow with the following parameters: sample QC Threshold; QC call_rate: ≥97, SNP QC Threshold; scr-cutoff: ≥95, and therefore, markers with high-resolution were analysed further. For imputation, we used TOPMed Imputation Server (https://imputation.biodatacatalyst.nhlbi.nih.gov/ ). To perform GWAS, imputed chromosome files were merged and VCF format files were further converted to plink format. The population stratification was performed using PLINK v1.9. GWAS analysis was performed using PLINK v1.9 [94 (link)] and SAIGE v 0.44.5 [95 (link)] at minor allele frequency (MAF) >0.05. Comparison between different groups was done as; deceased vs recovered, deceased vs asymptomatic and recovered vs asymptomatic patients.
6
Extraction and Quantification of Extracellular Vesicles and Bacterial DNA from Urine
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Each urine sample was centrifuged at 10,000× g for 10 min at 4 °C, and the supernatant was passed through a 0.22 μm membrane filter to eliminate any foreign particles and quantified based on protein concentration. Isolated EVs from each sample (1 μg of protein) were boiled at 100 °C for 40 min and centrifuged at 13,000× g for 30 min. Bacterial DNA was extracted from the collected supernatants using a DNA extraction kit (PowerSoil DNA Isolation Kit; MO BIO, Carlsbad, CA, USA) following the manufacturer’s instructions. Isolated DNA was quantified using the QIAxpert system (QIAGEN, Hilden, Germany).
7
Bacterial DNA Extraction from Stool EVs
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Human stool samples were diluted in 10 mL of PBS and filtered through a cell strainer. EVs were separated from the filtered samples by centrifuging the solution at 10,000×g for 10 min at 4 °C. The EVs were suspended in the supernatant of the samples, and the bacterial cells were concentrated in the pellets. The supernatants were then filtered through a 0.22-μm filter to eliminate bacterial and foreign particles from the stool samples. EVs were heated at 100 °C for 40 min to extract genomic material from the bacterial cells and EVs, and centrifuged for 30 min at 13,000 rpm at 4 °C to remove the remaining foreign particles and wastes. The DNeasy PowerSoil Kit (QIAGEN, Germany) was used to extract DNA from the samples, which was quantified using a QIAxpert system (QIAGEN, Germany).
8
Isolation and Characterization of Bacterial EVs
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Tumor tissues were frozen in liquid nitrogen, and the precooled tissues were ground using a steel bar. After grinding, 500 μL of phosphate-buffered saline (PBS) was added. The mixtures were homogenized by vortexing for 30 s. Each sample was centrifuged at 10,000× g for 10 min at 4 °C. The supernatant, comprised of EVs, was passed through a 0.22-μm membrane filter to eliminate foreign particles. The separated bacterial EVs were boiled at 100 °C for 40 min to eliminate the remaining floating particles and waste. They were then centrifuged at 13,000× g for 30 min at 4 °C. The supernatants were collected, and protein concentration was determined. Bacterial DNA was extracted from the prepared EVs using a DNA extraction kit (PowerSoil DNA Isolation Kit, MO BIO Laboratories, Solana Beach, CA, USA) following the manufacturer’s instructions. The isolated DNA was quantified using the QIAxpert system (QIAGEN, Hilden, Germany) [45 (link)].
9
Extraction and Quantification of EV DNA
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DNA extraction from EVs was performed as follows4 (link). The serum from each participant was collected in serum separator tubes and centrifuged at 3000 rpm for 15 min at 4 °C. The supernatant was collected and stored at − 80 °C until analysis. Then, the supernatant was mixed with 1 × phosphate-buffered saline (pH 7.4, ML008-01, Welgene, Republic of Korea) and subsequently centrifuged at 10,000× g for 10 min at 4 °C. To isolate EVs, bacteria and foreign particles were thoroughly eliminated through sterilization of the supernatant using a 0.22 μm filter. The separated EVs were boiled for 40 min at 100 °C and centrifuged at 13,000 rpm for 30 min at 4 °C to eliminate the remaining floating particles and waste. EV DNA was extracted using the DNeasy Powersoil Kit (QIAGEN, Germany) and quantified using the QIAxpert system (QIAGEN).
10
Isolation and Characterization of Extracellular Vesicles from Fecal and Cecal Samples
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Human feces and mouse cecal fluid samples were filtered through a cell strainer after being diluted in 10 mL of PBS for 24 hours. EVs contained in the stool samples were isolated by centrifugation at 10,000 × g for 10 min at 4°C. After centrifugation, the resulting bacterial cell pellet and EV-containing supernatant were separated. DNA contained within the bacterial pellet and supernatant was extracted using a DNA isolation kit (PowerSoil DNA Isolation Kit, MO BIO Laboratory, CA, USA) following the standard protocol in the kit guide. The DNA extracted from the isolated bacterial cells and EVs contained in each sample was quantified using a QIAxpert system (QIAGEN, Hilden, Germany).
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